Methods and compositions for treatment of disorders with follistatin polypeptides

ABSTRACT

The disclosure provides, in part, follistatin polypeptides that are suitable for use in local administration and methods for use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. application Ser. No.14/731,009 (now pending), which claims the benefit of priority to U.S.Provisional Application Ser. No. 62/007,908 (now expired), filed Jun. 4,2014. The specifications of the foregoing applications are herebyincorporated in their entirety.

BACKGROUND OF THE INVENTION

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. The family is dividedinto two general branches: the BMP/GDF and the TGF-beta/Activin/BMP10branches, whose members have diverse, often complementary effects. Bymanipulating the activity of a member of the TGF-beta family, it isoften possible to cause significant physiological changes in anorganism. For example, the Piedmontese and Belgian Blue cattle breedscarry a loss-of-function mutation in the GDF8 (also called myostatin)gene that causes a marked increase in muscle mass. Grobet et al., Nat.Genet. 1997, 17(1):71-4. Furthermore, in humans, inactive alleles ofGDF8 are associated with increased muscle mass and, reportedly,exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

Changes in muscle, bone, cartilage and other tissues may be achieved byagonizing or antagonizing signaling that is mediated by an appropriateTGF-beta family member. However, because members of the family mayaffect more than one tissue, it is desirable in some patient caresituations to achieve therapeutic inhibition of members of this familyin a localized, rather than systemic, manner. Thus, there is a need foragents that function as potent regulators of TGF-beta signaling and areappropriate for localized administration.

SUMMARY OF THE INVENTION

In part, the disclosure provides follistatin polypeptides that aredesigned to inhibit follistatin ligands (e.g. activin A, activin B, GDF8and GDF11) in proximity to the tissue in which such follistatinpolypeptides are administered, while having little or no systemiceffects on the patient.

Follistatin polypeptides described herein include polypeptidescomprising an amino acid sequence that is at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to any of SEQ ID NOS:1-4, 7-16 and 26-43. Optionally, the follistatin polypeptide is designedto dimerize or form higher order multimers. This may be achieved byfusing the follistatin sequence of a follistatin polypeptide to a domainthat confers dimerization or multimerization. An example of such adomain is a constant domain of an immunoglobulin, including, forexample, the Fc portion of an immunoglobulin. Optionally, thefollistatin portion is connected directly to the heterologous portion,or an intervening sequence such as a linker may be employed. An exampleof a linker is the sequence TGGG. Optionally the follistatin polypeptidemay exhibit heparin binding activity, in the manner that humanfollistatin-288 has heparin binding activity. Alternatively, thefollistatin may have a masked heparin binding domain, in the manner ofhuman follistatin-315. In part the disclosure provides therapeuticallyoptimized follistatin polypeptides that comprise a portion of animmunoglobulin constant domain from a human IgG that has reduced ADCC orCDC activity relative to native human IgG1. Examples include IgG2, IgG3,IgG4, hybrid IgG2/4 and variants of IgG1, IgG2, IgG3 and IgG4. In partthe disclosure provides an optimal active form of follistatin,comprising, consisting essentially of or consisting of the amino acidsequence of SEQ ID NO:15 or 16 that provides superior protein qualitiesand activity relative to the native FST(288) and FST(315) forms,particularly in the context of dimeric fusion proteins such asfollistatin-Fc proteins.

In certain aspects, the disclosure provides a polypeptide comprising afirst amino acid sequence and a second amino acid sequence, wherein thefirst amino acid sequence consists of an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 15 and 16, and wherein thesecond amino acid sequence comprises a constant domain of animmunoglobulin. Optionally, there is a linker polypeptide positionedbetween the first amino acid sequence and second amino acid sequence.Optionally the linker polypeptide comprises, consists essentially of, orconsists of the sequence TGGG. Optionally, the second amino acidsequence comprises, consists essentially of or consists of, a constantdomain of an IgG immunoglobulin. Optionally, the second amino acidsequence comprises, consists essentially of or consists of a constantdomain of an IgG immunoglobulin that has reduced ADCC activity relativeto human IgG1. Optionally, the second amino acid sequence comprises,consists essentially of or consists of a constant domain of an IgGimmunoglobulin that has reduced CDC activity relative to human IgG1.Optionally, the second amino acid sequence comprises, consistsessentially of or consists of a constant domain of an IgG immunoglobulinselected from the group: IgG1, IgG2 and IgG4. Optionally, the secondamino acid sequence comprises or consists of an Fc portion of animmunoglobulin, such as an IgG immunoglobulin, which may be animmunoglobulin that has reduced ADCC, CDC or both relative to humanIgG1, examples of which include IgG2, IgG4 and an IgG2/4 hybrid orvarious mutations of any of IgG1, IgG2, IgG3 or IgG4. In certain aspectsthe disclosure provides follistatin polypeptides that comprise, consistessentially of or consist of an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical tothe sequence selected from the group of SEQ ID NO: 38-43. In certainaspects the disclosure provides follistatin polypeptides that comprise,consist essentially of or consist of an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identicalto the sequence selected from the group of SEQ ID NO: 26-28 and 32-34.In certain aspects the disclosure provides follistatin polypeptides thatcomprise, consist essentially of or consist of an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to the sequence selected from the group of SEQ ID NO:29-31 and 35-37. Desirable follistatin polypeptides may bind to one ormore ligands selected from the group consisting of: myostatin, GDF-11,activin A and activin B with a KD less than 1 nM, 100 pM, 50 pM or 10pM. In certain aspects any of the above mentioned polypeptides may bedimers, including heterodimers or homodimers, or higher order multimers.Any of the above mentioned polypeptides may be incorporated into apharmaceutical preparation.

In certain aspects the disclosure provides nucleic acids encoding any ofthe follistatin polypeptides disclosed herein and cells comprising suchnucleic acids, which cells may be used to produce the follistatinpolypeptides.

In certain aspects, the disclosure provides methods for treating tissuesor organs by administering a follistatin polypeptide directly to suchtissue. For example, the disclosure provides a method of increasingmuscle size or strength in a patient, the method comprisingadministering an effective amount of a follistatin polypeptide by anintramuscular route of administration to a targeted muscle of a patientin need thereof, wherein the increased muscle size or strength occurs inthe targeted muscle, and wherein the follistatin polypeptide does nothave a substantial systemic effect on muscle size or strength. Thetargeted muscle may be damaged, weakened or deficient, as may be thecase in a variety of muscle disorders including muscular dystrophies(such as Duchenne muscular dystrophy, Becker's muscular dystrophy andfascioscapulohumeral muscular dystrophy), inflammatory muscle disorders(such as inclusion body myositis), muscle injury or trauma, muscledisuse (as may occur after prolonged bed rest or limb immobilization)and muscle atrophy or weakening as a consequence of aging, cancer orchronic diseases of various types. The methods may also be applied tomuscle that is healthy but for which an increase in muscle size orstrength of the targeted muscle is desired. Additionally, administrationof a follistatin polypeptide to muscle may cause a general decrease inbody fat and thereby be useful for treating obesity or other disordersassociated with excess body fat, and optionally, follistatin may beadministered directly to adipose tissue. The follistatin polypeptide maybe administered to only one targeted muscle or to more than one targetedmuscle. The methods and follistatin polypeptides may be used to achieveeffects on the targeted tissue, e.g. muscle, without substantial effectson other tissues, such as a non-targeted muscle or other organs. As aconsequence systemic effects of follistatin may not be observed. Forexample, a muscle that is contralateral to a targeted muscle may notsubstantially increase in size or strength or there may be nosubstantial effect in the patient on a measure selected from the groupconsisting of: serum FSH levels, liver size, hematocrit, hemoglobin andreticulocyte levels.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Patent Office upon request andpayment of the necessary fee.

FIG. 1 shows the full, unprocessed amino acid sequence of humanfollistatin 315 (SEQ ID NO:3). The leader sequence is italicized in boldfont, the follistatin N-terminal region (FSN) is indicated by singleunderlining, and the three follistatin domains (FSDs) are indicated bydouble underlining. In particular, follistatin domain I (FSDI) isindicated in red font, follistatin domain II (FSDII) is indicated inblue font, and the follistatin domain III (FSDIII) is indicated in greenfont.

FIG. 2 shows the effect of 4 weeks treatment, by subcutaneous injection,with either FST(288)-Fc, FST(315)-Fc, or ActRIIB-Fc on lean tissue massin mice. Vehicle was Tris-buffered saline. Data are means±SEM. *, P<0.05vs. TBS by unpaired t-test. #, P<0.05 vs. FST groups by unpaired t-test.FST(288)-Fc, FST(315)-Fc, and ActRIIB-Fc treatment resulted insignificant increases in lean tissue mass compared to vehicle controlmice. The increase in lean tissue mass of ActRIIB-Fc treated mice wassignificantly greater than the increases in lean tissue mass observed ineither FST(288)-Fc or FST(315)-Fc treated mice.

FIG. 3 shows the effect of 4 weeks treatment, by subcutaneous injectiontwice per week, with either FST(288)-Fc, FST(315)-Fc, or ActRIIB-Fc ongrip strength in mice. Vehicle was Tris-buffered saline. Data aremeans±SEM. *, P<0.05 vs. TBS by unpaired t-test. #, P<0.05 vs. FSTgroups by unpaired t-test. ActRIIB-Fc treatment increased grip strengthin mice. No increased grip strength was observed in FST(288)-Fc orFST(315)-Fc treated mice.

FIG. 4 shows the effect of 4 weeks treatment, by subcutaneous injectiontwice per week, with either FST(288)-IgG1, FST(315)-IgG1, or ActRIIB-Fcon pectoralis (Pecs), tibialis anterior (TA), gastrocnemius (Gastroc),and femoris muscle mass in mice. Vehicle was Tris-buffered saline. Dataare means±SEM. *, P<0.05 vs. TBS by unpaired t-test. #, P<0.05 vs. FSTgroups by unpaired t-test. ActRIIB-Fc treatment significantly increasedpectoralis, tibialis anterior, gastrocnemius, and femoris muscle mass inmice, but little to no increase in muscle mass was observed inFST(288)-IgG1 or FST(315)-IgG1 treated mice.

FIG. 5 shows the effect of 4 weeks treatment, by subcutaneous injection,with either FST(288)-IgG1 or FST(315)-IgG1 on serum levels offollicle-stimulating hormone (FSH). Vehicle was Tris-buffered saline.Data are means±SEM. *, P<0.05 vs. TBS by unpaired t-test. FST(315)-IgG1treatment resulted in a significant decrease in serum FSH levels incomparison to vehicle control mice. In contrast, FST(288)-IgG1 treatmenthad no effect on serum FSH levels

FIG. 6 shows the effect of 4 weeks treatment, by subcutaneous injectiontwice weekly, with either FST(288)-IgG1, FST(315)-IgG1, or ActRIIB-mFcon lean tissue mass in mice. Vehicle was Tris-buffered saline. Data aremeans±SEM. *, P<0.05 vs. TBS by unpaired t-test. ActRIIB-mFc treatmentresulted in significant increases in lean tissue mass compared tovehicle control mice. No increases in lean tissue mass were observed ineither FST(288)-IgG1 or FST(315)-IgG1 treated mice.

FIG. 7 shows the effect of 4 weeks treatment, by intramuscular injectioninto the right gastrocnemius twice weekly, with either FST(288)-IgG1,FST(315)-IgG1, or ActRIIB-mFc on gastrocnemius muscle mass in mice.Vehicle was Tris-buffered saline. Data are means±SEM. *, P<0.05 vs. TBSby unpaired t-test. #, P<0.05 right, injected gastocnemius muscle vs.left, non-injected, gastocnemius muscle by unpaired t-test.FST(288)-IgG1, FST(315)-IgG1, and ActRIIB-mFc treatment significantlyincreased muscle mass in the right, injected gastocnemius muscle.ActRIIB-mFc treatment also significantly increased muscle mass in theleft, non-injected gastocnemius muscle. In contrast, there was notobserved increase in the left, non-injected gastocnemius muscle inFST(288)-IgG1 or FST(315)-IgG1 treated mice.

FIG. 8 shows the effect of 3 weeks treatment, by intramuscular injectioninto the right gastrocnemius twice weekly, with varying doses ofFST(288)-IgG1, on gastrocnemius muscle mass in mice, expressed as aratio over the uninjected, left gastrocnemius. Vehicle wasphosphate-buffered saline. Data are means±SEM. *, P<0.05 vs. PBS byunpaired t-test. Increasing doses of FST(288)-IgG1 caused an increasinghypertrophy of the injected gastocnemius muscle relative to theuninjected muscle.

FIG. 9 shows the effect of 4 weeks treatment, by intramuscular injectioninto the left gastrocnemius twice weekly, with FST(291)-IgG1. Vehiclewas phosphate-buffered saline. Data are means±SEM. *, P<0.05 vs. PBS byunpaired t-test. Intramuscular administration of FST(291)-IgG2 causedmarked increase in muscle mass in the injected gastocnemius musclerelative to the uninjected muscle and relative to controls.

DETAILED DESCRIPTION 1. Overview

In certain aspects, the present disclosure relates to follistatinpolypeptides. As used herein, the term “follistatin” refers to a familyof follistatin (FST) proteins and follistatin-related proteins, derivedfrom any species. Follistatin is an autocrine glycoprotein that isexpressed in nearly all tissues of higher animals. It was initiallyisolated from follicular fluid and was identified as a protein fractionthat inhibited follicle-stimulating hormone (FSH) secretion from theanterior pituitary, and therefore was designated as FSH-suppressingprotein (FSP). Subsequently, its primary function has been determined tobe the binding and neutralization of members of the TGF-β superfamilyincluding, for example, activin, a paracrine hormone that enhancessecretion of FSH in the anterior pituitary.

The term “follistatin polypeptide” is used to refer to polypeptidescomprising any naturally occurring polypeptide of the follistatin familyas well as any variants thereof (including mutants, fragments, fusions,and peptidomimetic forms) that retain a useful activity, including, forexample, ligand binding (e.g., myostatin, GDF-11, activin A, activin B)or heparin binding. For example, follistatin polypeptides includepolypeptides comprising an amino acid sequence derived from the sequenceof any known follistatin having a sequence at least about 80% identicalto the sequence of a follistatin polypeptide, and preferably at least85%, 90%, 95%, 97%, 99% or greater identity. The term “follistatinpolypeptide” may refer to fusion proteins that comprise any of thepolypeptides mentioned above along with a heterologous (non-follistatin)portion. An amino acid sequence is understood to be heterologous tofollistatin if it is not uniquely found in the long (315 amino acid)form of human follistatin, represented by SEQ ID NO:3. Many examples ofheterologous portions are provided herein, and such heterologousportions may be immediately adjacent, by amino acid sequence, to thefollistatin polypeptide portion of a fusion protein, or separated byintervening amino acid sequence, such as a linker or other sequence.

Follistatin is a single-chain polypeptide with a range of molecularweights from 31 to 49 kDa based on alternative mRNA splicing andvariable glycosylation of the protein. The alternatively spliced mRNAsencode two proteins of 315 amino acids (i.e., FST315) and 288 aminoacids (i.e., FST288); follistatin 315 can be further proteolyticallydegraded to follistatin 303 (FST303). Analysis of the amino acidsequence has revealed that the native human follistatin polypeptidecomprises five domains (from the N-terminal side): a signal sequencepeptide (amino acids 1-29 of SEQ ID NO:1), an N-terminal domain (FSN)(amino acids 30-94 of SEQ ID NO:1), follistatin domain I (FSDI) (aminoacids 95-164 of SEQ ID NO:1), follistatin domain II (FSDII) (amino acids(168-239 of SEQ ID NO:1), and follistatin domain III (FSDIII) (aminoacids 245-316 of SEQ ID NO:1). See PNAS, U.S.A., 1988, Vol. 85, No 12,pp 4218-4222.

The human follistatin-288 (FST288) precursor has the following aminoacid sequence, with the signal peptide indicated in bold, the N-terminaldomain (FSN) indicated by single underlining, and the follistatindomains I-III (FSI, FSII, FSIII) indicated by double underlining.

(SEQ ID NO: 1) MVRARHQPGGLCLLLLLLCQFMEDRSQA GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKET CENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAA CSSGVLLEVKHSGSCN

The processed (mature) human follistatin variant FST(288) has thefollowing amino acid sequence with the N-terminal domain indicated bysingle underlining, and the follistatin domains I-III indicated bydouble underlining. Moreover, it will be appreciated that any of theinitial amino acids G or N, prior to the first cysteine may be removedby processing or intentionally eliminated without any consequence, andpolypeptides comprising such slightly smaller polypeptides are furtherincluded.

(SEQ ID NO: 2) GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDTLFKWMIFNGGAPNCIPCKET CENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCN

The human follistatin-315 (FST315) precursor has the following aminoacid sequence, with the signal peptide indicated in bold, the N-terminaldomain (FSN) indicated by single underlining, and the follistatindomains I-III (FSI, FSII, FSIII) indicated by double underlining (NCBIAccession Number AAH04107.1; 344 amino acids).

(SEQ ID NO: 3) MVRARHQPGGLCLLLLLLCQFMEDRSAQA GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKET CENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEW

The processed (mature) human FST(315) has the following amino acidsequence with the N-terminal domain indicated by single underlining, andthe follistatin domains I-III indicated by double underlining. Moreover,it will be appreciated that any of the initial amino acids G or N, priorto the first cysteine may be removed by processing or intentionallyeliminated without any consequence, and polypeptides comprising suchslightly smaller polypeptides are further included.

(SEQ ID NO: 4) GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKET CENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEED EDQDYSFPISSILEW

Follistatin polypeptides of the disclosure may include any naturallyoccurring domain of a follistatin protein as well as variants thereof(e.g., mutants, fragments, and peptidomimetic forms) that retain auseful activity. For example, it is well-known that FST(315) andFST(288) have high affinity for both activin (activin A and activin B)and myostatin (and the closely related GDF11) and that the follistatindomains (e.g., FSN and FSD I-III) are thought to be involved in thebinding of such TGF-β ligands. However, it believed that each of thesethree domains may have a different affinity for these TGF-β ligands. Forexample, a recent study has demonstrated that polypeptide constructscomprising only the N-terminal domain (FSN) and two FSDI domains intandem retained high affinity for myostatin, demonstrated little or noaffinity for activin and promoted systemic muscle growth when introducedinto a mouse by gene expression (Nakatani et al., The FASEB Journal,Vol. 22477-487 (2008)).

Additionally, the FSDI domain contains the heparin binding domain ofhuman follistatin, which has the amino acid sequence of KKCRIVINKKNKPR(SEQ ID NO: 5). This heparin binding domain can be represented asBBXBXXBBXBXB (SEQ ID NO:6) wherein “B” means a basic amino acid,particularly lysine (K) or arginine (R). Accordingly, the presentdisclosure encompasses, in part, variant follistatin proteins thatdemonstrate selective binding and/or inhibition of a given TGF-β ligandrelative to the naturally occurring FST protein (e.g., maintaininghigh-affinity for myostain while having a significantly reduced affinityfor activin).

In certain aspects, the disclosure includes polypeptides comprising theFSN domain, as set forth below, and, for example, one or moreheterologous polypeptide, and moreover, it will be appreciated that anyof the initial amino acids G or N, prior to the first cysteine may bedeleted, as in the example shown below (SEQ ID NO:8).

(SEQ ID NO: 7) GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKET (SEQ ID NO: 8)CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIF NGGAPNCIPCKET

In certain aspects, the disclosure includes polypeptides comprising theFSDI domain which contains the minimal core activities of myostatin(and/or GDF11) binding along with heparin binding as set forth below,and, for example, one or more heterologous polypeptide.

(SEQ ID NO: 9) CENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRC

An FSDI sequence may be advantageously maintained in structural contextby expression as a polypeptide further comprising the FSN domain.Accordingly, the disclosure includes polypeptides comprising theFSN-FSDI sequence, as set forth below (SEQ ID NO:10), and, for example,one or more heterologous polypeptide, and moreover, it will beappreciated that any of the initial amino acids G or N, prior to thefirst cysteine may be removed by processing or intentionally eliminatedwithout any consequence, and polypeptides comprising such slightlysmaller polypeptides are further included.

(SEQ ID NO: 10) CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRC

As demonstrated by Nakani et al., an FSN-FSDI-FSDI construct issufficient to confer systemic muscle growth when genetically expressedin a mouse, and accordingly the disclosure includes polypeptidescomprising the amino acid sequences below and, for example, one or moreheterologous polypeptide.

(SEQ ID NO: 11) CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCENVDCGPGKKCRMNKKNKPRCVCAPDCSNTIWKGPVCGLDGKTYRNECALLKARCKEQPELEV QYQGRC

While the FSDI sequence confers myostatin and GDF11 binding, it has beendemonstrated that activins, particularly Activin A but also Activin B,are also negative regulators of muscle, and therefore a follistatinpolypeptide that inhibits both the myostatin/GDF11 group and the activinA/activin B group may provide a more potent muscle effect. Moreover, inview of the findings herein demonstrating the low systemic availabilityof certain follistatin polypeptides, particularly those comprising aheparin binding domain, and more particularly in a homodimeric form,such as an Fc fusion, safety concerns associated with the known effectsof activin inhibition on the reproductive axis and other tissues arealleviated. Given that FSDII confers activin A and B binding, thedisclosure provides polypeptides comprising FSDI and FSDII (SEQ IDNO:12), as well as FSN-FSDI-FSDII constructs (SEQ ID NOS: 13) and, forexample, one or more heterologous polypeptide.

(SEQ ID NO: 12) CENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKC (SEQ ID NO: 13)CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSI GLAYEGKC

As described in the Examples, a follistatin polypeptide of 291 aminoacids (representing a truncation of the naturally occurring FST-315) hasadvantageous properties. Accordingly, unprocessed (SEQ ID NO: 14) andmature FST(291) (SEQ ID NO: 15) polypeptides are included in thedisclosure and may be combined with heterologous proteins. Moreover, itwill be appreciated that any of the initial amino acids G or N, prior tothe first cysteine may be removed by processing or intentionallyeliminated without any consequence, and polypeptides comprising suchslightly smaller polypeptides are further included, such as the exampleshown below (SEQ ID NO:16).

(SEQ ID NO: 14) MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEA ACSSGVLLEVKHSGSCNSIS(SEQ ID NO: 15) GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSIS (SEQ ID NO: 16)CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSIS

In certain embodiments, the present invention relates to antagonizing aligand of follistatin (also referred to as a follistatin ligand) with asubject follistatin polypeptide (e.g., an FST-IgG fusion polypeptide).Thus, compositions and methods of the present disclosure are useful fortreating disorders associated with abnormal activity of one or moreligands of follistatin. Exemplary ligands of follistatin include someTGF-β family members, such as activin A, activin B, myostatin (GDF8) andGDF11.

Follistatin proteins herein may be referred to as FST. If followed by anumber, such as FST(288), this indicates that the protein is the 288form of follistatin. If presented as FST(288)-Fc, this indicates aC-terminal Fc fusion to the FST(288), which may or may not include anintervening linker. The Fc in this instance may be any immunoglobulin Fcportion as that term is defined herein. If presented as FST(288)-IgG2,this indicates a C-terminal Fc fusion to the FST(288) of the Fc portionof human IgG2.

Activins are dimeric polypeptide growth factors and belong to the TGF-βsuperfamily. There are three activins (A, B, and AB) that arehomo/heterodimers of two closely related β subunits (β_(A)β_(A),β_(B)β_(B), and β_(A)β_(B)). Additional activins C and E have beenidentified, although the function of these proteins is poorlyunderstood. In the TGF-β superfamily, activins are unique andmultifunctional factors that can stimulate hormone production in ovarianand placental cells, support neuronal cell survival, influencecell-cycle progress positively or negatively depending on cell type, andinduce mesodermal differentiation at least in amphibian embryos (DePaoloet al., 1991, Proc SocEp Biol Med. 198:500-512; Dyson et al., 1997, CurrBiol. 7:81-84; Woodruff, 1998, Biochem Pharmacol. 55:953-963). Moreover,erythroid differentiation factor (EDF) isolated from the stimulatedhuman monocytic leukemic cells was found to be identical to activin A(Murata et al., 1988, PNAS, 85:2434). It was suggested that activin Aacts as a natural regulator of erythropoiesis in the bone marrow. Inseveral tissues, activin signaling is antagonized by its relatedheterodimer, inhibin. For example, during the release offollicle-stimulating hormone (FSH) from the pituitary, activin promotesFSH secretion and synthesis, while inhibin prevents FSH secretion andsynthesis. Activin has also been implicated as a negative regulator ofmuscle mass and function, and activin antagonists can promote musclegrowth or counteract muscle loss in vivo. Link and Nishi, Exp Cell Res.1997 Jun. 15; 233(2):350-62; He et al., Anat Embryol (Berl). 2005 June;209(5):401-7; Souza et al. Mol Endocrinol. 2008 December;22(12):2689-702; Am J Physiol Endocrinol Metab. 2009 July;297(1):E157-64; Gilson et al. Zhou et al. Cell. 2010 Aug. 20;142(4):531-43.

Growth and Differentiation Factor-8 (GDF8) is also known as myostatin.GDF8 is a negative regulator of skeletal muscle mass. GDF8 is highlyexpressed in the developing and adult skeletal muscle. The GDF8 nullmutation in transgenic mice is characterized by a marked hypertrophy andhyperplasia of the skeletal muscle (McPherron et al., Nature, 1997,387:83-90). Similar increases in skeletal muscle mass are evident innaturally occurring mutations of GDF8 in cattle (Ashmore et al., 1974,Growth, 38:501-507; Swatland and Kieffer, J. Anim. Sci., 1994,38:752-757; McPherron and Lee, Proc. Natl. Acad. Sci. USA, 1997,94:12457-12461; and Kambadur et al., Genome Res., 1997, 7:910-915) and,strikingly, in humans (Schuelke et al., N Engl J Med 2004; 350:2682-8).Studies have also shown that muscle wasting associated withHIV-infection in humans is accompanied by increases in GDF8 proteinexpression (Gonzalez-Cadavid et al., PNAS, 1998, 95:14938-43). Inaddition, GDF8 can modulate the production of muscle-specific enzymes(e.g., creatine kinase) and modulate myoblast cell proliferation (WO00/43781). The GDF8 propeptide can noncovalently bind to the mature GDF8domain dimer, inactivating its biological activity (Miyazono et al.(1988) J. Biol. Chem., 263: 6407-6415; Wakefield et al. (1988) J. Biol.Chem., 263; 7646-7654; and Brown et al. (1990) Growth Factors, 3:35-43). Other proteins which bind to GDF8 or structurally relatedproteins and inhibit their biological activity include follistatin, andpotentially, follistatin-related proteins (Gamer et al. (1999) Dev.Biol., 208: 222-232).

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants(sequence variants). Such comparisons typically comprise alignments ofpolymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions. However, in common usage andin the instant application, the term “homologous,” when modified with anadverb such as “highly,” may refer to sequence similarity and may or maynot relate to a common evolutionary origin.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

2. Follistatin Polypeptides

In certain aspects, the disclosure relates to follistatin polypeptides(e.g., FST-Fc polypeptides), and particularly truncated formsexemplified by polypeptides comprising SEQ ID NO:2, 7, 8, 9, 10, 11, 12,13, 14, 15 or 16, and variants thereof. Optionally, the fragments,functional variants, and modified forms have similar, the same orimproved biological activities of their corresponding wild-typefollistatin polypeptides. For example, a follistatin variant of thedisclosure may bind to and inhibit function of a follistatin ligand(e.g., activin A, activin AB, activin B, and GDF8). Optionally, afollistatin polypeptide modulates growth of tissues, particularlymuscle. Examples of follistatin polypeptides include polypeptidescomprising, consisting essentially of or consisting of the amino acidsequences by any of SEQ ID Nos. 1-16 and 26-43, as well as polypeptidescomprising, consisting essentially of or consisting of amino acidsequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identical to an amino acid sequence of any of SEQID Nos. 1-16 and 26-43. Variations on these polypeptides may be preparedaccording to the following guidance. The numbering of amino acids in thefollistatin polypeptides is based on the sequence of SEQ ID NO:1,regardless of whether the native leader sequence is used.

As described above, follistatin is characterized by three cysteine-richregions (i.e., FS domains I-III) that are believed to mediatefollistatin-ligand binding. Furthermore, researchers have demonstratedthat polypeptide constructs comprising only one of the three FS-bindingdomains (e.g., FSDI) retains strong affinity towards certainfollistatin-ligands (e.g., myostatin) and is biologically active invivo. See Nakatani et al., The FASEB Journal, Vol. 22477-487 (2008).Therefore, variant follistatin polypeptides of the disclosure maycomprise one or more active portions of a follistatin protein. Forexample, constructs of the disclosure may begin at a residuecorresponding to amino acids 30-95 of SEQ ID NO:1 and end at a positioncorresponding to amino acids 316-344 of SEQ ID NO:1. Other examplesinclude constructs that begin at a position from 30-95 of SEQ ID NO:1and end at a position corresponding to amino acids 164-167 or 238-244.Others may include any of SEQ ID Nos. 7-16.

The follistatin variations described herein may be combined in variousways with each other or with heterologous amino acid sequences. Forexample, variant follistatin proteins of the disclosure includepolypeptides that comprise one or more FS domains selected from FSDI(amino acids 95-164 of SEQ ID NO:1 (i.e., SEQ ID NO:2), FSDII (aminoacids 168-239 of SEQ ID NO:1), or FSDIII (amino acids 245-316 of SEQ IDNO:1) as well as proteins that comprise one or more FS domains selectedfrom a sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%identical to FSDI (amino acids 95-164 of SEQ ID NO:1 (i.e., SEQ IDNO:2), FSDII (amino acids 168-239 of SEQ ID NO:1), or FSDIII (aminoacids 245-316 of SEQ ID NO:1). These FS domains may be combined in anyorder within a variant follistatin polypeptide of the disclosureprovided that such recombinant proteins maintain the desired activityincluding, for example, follistatin ligand-binding activity (e.g.,myostatin) and biological activity (e.g., inducing muscle mass and/orstrength). Examples of such follistatin variant polypeptides include,for example, polypeptides having domain structures such asFSDI-FSDII-FSDIII, FSDI-FSDIII, FSDI-FSDI-FSDIII, FSDI-FSDII, FSDI-FSDI,FSN-FSDI-FSDII-FSDIII, FSN-FSDI-FSDII, FSN-FSDI-FSDI, FSN-FSDI-FSDIII,FSN-FSDI-FSDI-FSDIII, and polypeptides obtained by fusing otherheterologous polypeptides to the N-termini or the C-termini of thesepolypeptides. These domains may be directly linked or liked via a linkerpolypeptide. Optionally, polypeptide linkers may be any sequence and maycomprise 1-50, preferably 1-10, and more preferably 1-5 amino acids. Incertain aspects, preferred linkers contain no cysteine amino acids.

In some embodiments, follistatin variants of the disclosure have reducedor abolished binding affinity for one or more follistatin ligands. Incertain aspects, the disclosure provides follistatin variants that havereduced or abolished binding affinity for activin. In certain aspects,the disclosure provides follistatin variants that have reduced orabolished binding affinity for activin but retain high affinity formyostatin.

In certain aspects, the disclosure provides follistatin variants that donot comprise a sequence corresponding to the FSDII domain orfunctionally active FSDII domain. For example, follistatin polypeptidesof the disclosure may include a variant obtained through partial orcomplete deletion of the FSDII domain. In certain aspects, suchfollistatin variants include the deletion of one or more cysteineresidues within the FSDII region or substitution with non-cysteine aminoacids.

The follistatin proteins of the disclosure may comprise a signalsequence. The signal sequence can be a native signal sequence of afollistatin protein (e.g., amino acids 1-29 of SEQ ID NO:1) or a signalsequence from another protein, such as tissue plasminogen activator(TPA) signal sequence or a honey bee melatin (HBM) signal sequence.

Further N-linked glycosylation sites (N-X-S/T) may be added to afollistatin polypeptide, and may increase the serum half-life of anFST-Fc fusion protein. N-X-S/T sequences may be generally introduced atpositions outside the ligand-binding pocket. N—X-S/T sequences may beintroduced into the linker between the follistatin sequence and the Fcor other fusion component. Such a site may be introduced with minimaleffort by introducing an N in the correct position with respect to apre-existing S or T, or by introducing an S or T at a positioncorresponding to a pre-existing N. Any S that is predicted to beglycosylated may be altered to a T without creating an immunogenic site,because of the protection afforded by the glycosylation. Likewise, any Tthat is predicted to be glycosylated may be altered to an S.Accordingly, a follistatin variant may include one or more additional,non-endogenous N-linked glycosylation consensus sequences.

In certain embodiments, the present disclosure contemplates makingfunctional variants by modifying the structure of a follistatinpolypeptide for such purposes as enhancing therapeutic efficacy, orstability (e.g., ex vivo shelf life and resistance to proteolyticdegradation in vivo). Modified follistatin polypeptides can also beproduced, for instance, by amino acid substitution, deletion, oraddition. For instance, it is reasonable to expect that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid (e.g., conservativemutations) will not have a major effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Whether a change in the amino acid sequence of a follistatinpolypeptide results in a functional homolog can be readily determined byassessing the ability of the variant follistatin polypeptide to producea response in cells in a fashion similar to the wild-type follistatinpolypeptide, or to bind to one or more ligands, such as activin ormyostatin in a fashion similar to wild-type follistatin.

In certain embodiments, the present invention contemplates specificmutations of the follistatin polypeptides so as to alter theglycosylation of the polypeptide. Such mutations may be selected so asto introduce or eliminate one or more glycosylation sites, such asO-linked or N-linked glycosylation sites. Asparagine-linkedglycosylation recognition sites generally comprise a tripeptidesequence, asparagine-X-threonine (where “X” is any amino acid) which isspecifically recognized by appropriate cellular glycosylation enzymes.The alteration may also be made by the addition of, or substitution by,one or more serine or threonine residues to the sequence of thewild-type follistatin polypeptide (for O-linked glycosylation sites). Avariety of amino acid substitutions or deletions at one or both of thefirst or third amino acid positions of a glycosylation recognition site(and/or amino acid deletion at the second position) results innon-glycosylation at the modified tripeptide sequence. Another means ofincreasing the number of carbohydrate moieties on a follistatinpolypeptide is by chemical or enzymatic coupling of glycosides to thefollistatin polypeptide. Depending on the coupling mode used, thesugar(s) may be attached to (a) arginine and histidine; (b) freecarboxyl groups; (c) free sulfhydryl groups such as those of cysteine;(d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline; (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan; or (f) the amide group of glutamine. Thesemethods are described in WO 87/05330 published Sep. 11, 1987, and inAplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 259-306,incorporated by reference herein. Removal of one or more carbohydratemoieties present on an ActRIIB polypeptide may be accomplishedchemically and/or enzymatically. Chemical deglycosylation may involve,for example, exposure of the follistatin polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Chemical deglycosylation is further described byHakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge etal. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydratemoieties on follistatin polypeptides can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.(1987) Meth. Enzymol. 138:350. The sequence of a follistatin polypeptidemay be adjusted, as appropriate, depending on the type of expressionsystem used, as mammalian, yeast, insect and plant cells may allintroduce differing glycosylation patterns that can be affected by theamino acid sequence of the peptide. In general, follistatin proteins foruse in humans will be expressed in a mammalian cell line that providesproper glycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines are expected to be useful as well.

This disclosure further contemplates a method of generating variants,particularly sets of combinatorial variants of an follistatinpolypeptide, including, optionally, truncation variants; pools ofcombinatorial mutants are especially useful for identifying functionalvariant sequences. The purpose of screening such combinatorial librariesmay be to generate, for example, follistatin polypeptide variants thathave altered properties, such as altered pharmacokinetics, or alteredligand binding. A variety of screening assays are provided below, andsuch assays may be used to evaluate variants. For example, a follistatinpolypeptide variant may be screened for ability to bind to a follistatinpolypeptide, to prevent binding of a follistatin ligand to a follistatinpolypeptide.

The activity of a follistatin polypeptide or its variants may also betested in a cell-based or in vivo assay. For example, the effect of afollistatin polypeptide variant on the expression of genes involved inmuscle production may be assessed. This may, as needed, be performed inthe presence of one or more recombinant follistatin ligand proteins(e.g., activin A), and cells may be transfected so as to produce afollistatin polypeptide and/or variants thereof, and optionally, afollistatin ligand. Likewise, a follistatin polypeptide may beadministered to a mouse or other animal, and one or more muscleproperties, such as muscle mass or strength may be assessed. Such assaysare well known and routine in the art. A responsive reporter gene may beused in such cell lines to monitor effects on downstream signaling.

Combinatorially-derived variants can be generated which have a selectivepotency relative to a naturally occurring follistatin polypeptide. Suchvariant proteins, when expressed from recombinant DNA constructs, can beused in gene therapy protocols. Likewise, mutagenesis can give rise tovariants which have intracellular half-lives dramatically different thanthe corresponding a wild-type follistatin polypeptide. For example, thealtered protein can be rendered either more stable or less stable toproteolytic degradation or other processes which result in destructionof, or otherwise inactivation of a native follistatin polypeptide. Suchvariants, and the genes which encode them, can be utilized to alterfollistatin polypeptide levels by modulating the half-life of thefollistatin polypeptides. For instance, a short half-life can give riseto more transient biological effects and, when part of an inducibleexpression system, can allow tighter control of recombinant follistatinpolypeptide levels within the cell.

In certain embodiments, the follistatin polypeptides of the disclosuremay further comprise post-translational modifications in addition to anythat are naturally present in the follistatin polypeptides. Suchmodifications include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. As a result, the modified follistatin polypeptides maycontain non-amino acid elements, such as polyethylene glycols, lipids,poly- or mono-saccharide, and phosphates. Effects of such non-amino acidelements on the functionality of a follistatin polypeptide may be testedas described herein for other follistatin polypeptide variants. When afollistatin polypeptide is produced in cells by cleaving a nascent formof the follistatin polypeptide, post-translational processing may alsobe important for correct folding and/or function of the protein.Different cells (such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293)have specific cellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the follistatin polypeptides.

In certain aspects, functional variants or modified forms of thefollistatin polypeptides include fusion proteins having at least aportion of a follistatin polypeptide and one or more fusion domains.Well known examples of such fusion domains include, but are not limitedto, polyhistidine, Glu-Glu, glutathione S transferase (GST),thioredoxin, protein A, protein G, an immunoglobulin heavy chainconstant region (e.g., an Fc), maltose binding protein (MBP), or humanserum albumin. A fusion domain may be selected so as to confer a desiredproperty. For example, some fusion domains are particularly useful forisolation of the fusion proteins by affinity chromatography. For thepurpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- orcobalt-conjugated resins are used. Many of such matrices are availablein “kit” form, such as the Pharmacia GST purification system and theQIAexpress™ system (Qiagen) useful with (HIS₆) fusion partners. Asanother example, a fusion domain may be selected so as to facilitatedetection of the follistatin polypeptides. Examples of such detectiondomains include the various fluorescent proteins (e.g., GFP) as well as“epitope tags,” which are usually short peptide sequences for which aspecific antibody is available. Well known epitope tags for whichspecific monoclonal antibodies are readily available include FLAG,influenza virus haemagglutinin (HA), and c-myc tags. In some cases, thefusion domains have a protease cleavage site, such as for Factor Xa orThrombin, which allows the relevant protease to partially digest thefusion proteins and thereby liberate the recombinant proteins therefrom.The liberated proteins can then be isolated from the fusion domain bysubsequent chromatographic separation. In certain preferred embodiments,a follistatin polypeptide is fused with a domain that stabilizes thefollistatin polypeptide in vivo (a “stabilizer” domain). By“stabilizing” is meant anything that increases serum half-life,regardless of whether this is because of decreased destruction,decreased clearance by the kidney, or other pharmacokinetic effect.Fusions with the Fc portion of an immunoglobulin are known to conferdesirable pharmacokinetic properties on a wide range of proteins.Likewise, fusions to human serum albumin can confer desirableproperties. Other types of fusion domains that may be selected includemultimerizing (e.g., dimerizing, tetramerizing) domains and functionaldomains (that confer an additional biological function, such as furtherstimulation of muscle growth).

As specific examples, the present disclosure provides fusion proteinscomprising follistatin polypeptides fused to a polypeptide comprising aconstant domain of an immunoglobulin, such as a CH1 CH2 or CH3 domain ofan immunoglobulin or an Fc. Fc domains derived from human IgG1 and IgG2are provided below (SEQ ID NO: 17 and SEQ ID NO:18, respectively). Asdescribed herein, an IgG2, IgG4 or IgG2/4 Fc domain is particularlyadvantageous for fusion with follistatin polypeptides that retainheparin binding activity because these Fc species have reduced CDCand/or ADCC activity which may be harmful to the cells to which theseheparin binding polypeptides may adhere. Other mutations are known thatdecrease either CDC or ADCC activity, and collectively, any of thesevariants are included in the disclosure and may be used as advantageouscomponents of a follistatin fusion protein. Optionally, the Fc domain ofSEQ ID NO:17 has one or more mutations at residues such as Asp-265,Lys-322, and Asn-434 (numbered in accordance with the correspondingfull-length IgG1). In certain cases, the mutant Fc domain having one ormore of these mutations (e.g., Asp-265 mutation) has reduced ability ofbinding to the Fcγ receptor relative to a wildtype Fc domain. In othercases, the mutant Fc domain having one or more of these mutations (e.g.,Asn-434 mutation) has increased ability of binding to the MHC classI-related Fc-receptor (FcRN) relative to a wildtype Fc domain.

Examples of human IgG1 and IgG2 amino acid sequences that may beemployed are shown below:

IgG1 (SEQ ID NO: 17) THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG2 (SEQ ID NO: 18)VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, a follistatin polypeptide may be placedC-terminal to a heterologous domain, or, alternatively, a heterologousdomain may be placed C-terminal to a follistatin polypeptide. Thefollistatin polypeptide domain and the heterologous domain need not beadjacent in a fusion protein, and additional domains or amino acidsequences may be included C- or N-terminal to either domain or betweenthe domains.

As used herein, the term “immunoglobulin Fc domain” or simply “Fc” isunderstood to mean the carboxyl-terminal portion of an immunoglobulinchain constant region, preferably an immunoglobulin heavy chain constantregion, or a portion thereof. For example, an immunoglobulin Fc regionmay comprise 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2domain and a CH3 domain, or 5) a combination of two or more domains andan immunoglobulin hinge region. In a preferred embodiment theimmunoglobulin Fc region comprises at least an immunoglobulin hingeregion a CH2 domain and a CH3 domain, and preferably lacks the CH1domain. It is also understood that a follistatin polypeptide maycomprise only a domain of an immunoglobulin, such as a CH1 domain, a CH2domain or a CH3 domain. Many of these domains confer desirablepharmacokinetic properties as well as dimerization or higher ordermultimerization.

In one embodiment, the class of immunoglobulin from which the heavychain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or4). Other classes of immunoglobulin, IgA (Igα), IgD (Igδ), IgE (Igε) andIgM (Igμ), may be used. The choice of appropriate immunoglobulin heavychain constant region is discussed in detail in U.S. Pat. Nos. 5,541,087and 5,726,044. The choice of particular immunoglobulin heavy chainconstant region sequences from certain immunoglobulin classes andsubclasses to achieve a particular result is considered to be within thelevel of skill in the art. The portion of the DNA construct encoding theimmunoglobulin Fc region preferably comprises at least a portion of ahinge domain, and preferably at least a portion of a CH₃ domain of Fcgamma or the homologous domains in any of IgA, IgD, IgE, or IgM.

Furthermore, it is contemplated that substitution or deletion of aminoacids within the immunoglobulin heavy chain constant regions may beuseful in the practice of the methods and compositions disclosed herein.One example would be to introduce amino acid substitutions in the upperCH2 region to create an Fc variant with reduced affinity for Fcreceptors (Cole et al. (1997) J. Immunol. 159:3613). Additionally, inmany instances, the C-terminal lysine, or K, will be removed and thusany of the polypeptides described herein may omit the C-terminal K thatis found in an Fc domain, such as those shown in SEQ ID NO: 17 or SEQ IDNO: 18.

In certain embodiments, the follistatin polypeptides of the presentdisclosure contain one or more modifications that are capable ofstabilizing the follistatin polypeptides. For example, suchmodifications enhance the in vitro half-life of the follistatinpolypeptides, enhance circulatory half-life of the follistatinpolypeptides or reducing proteolytic degradation of the follistatinpolypeptides. Such stabilizing modifications include, but are notlimited to, fusion proteins (including, for example, fusion proteinscomprising a follistatin polypeptide and a stabilizer domain),modifications of a glycosylation site (including, for example, additionof a glycosylation site to a follistatin polypeptide), and modificationsof carbohydrate moiety (including, for example, removal of carbohydratemoieties from a follistatin polypeptide). In the case of fusionproteins, a follistatin polypeptide is fused to a stabilizer domain suchas an IgG molecule (e.g., an Fc domain). As used herein, the term“stabilizer domain” not only refers to a fusion domain (e.g., Fc) as inthe case of fusion proteins, but also includes nonproteinaceousmodifications such as a carbohydrate moiety, or nonproteinaceouspolymer, such as polyethylene glycol.

In certain embodiments, the present invention makes available isolatedand/or purified forms of the follistatin polypeptides, which areisolated from, or otherwise substantially free of, other proteins.

In certain embodiments, follistatin polypeptides (unmodified ormodified) of the disclosure can be produced by a variety of art-knowntechniques. For example, such follistatin polypeptides can besynthesized using standard protein chemistry techniques such as thosedescribed in Bodansky, M. Principles of Peptide Synthesis, SpringerVerlag, Berlin (1993) and Grant G. A. (ed.), Synthetic Peptides: AUser's Guide, W. H. Freeman and Company, New York (1992). In addition,automated peptide synthesizers are commercially available (e.g.,Advanced ChemTech Model 396; Milligen/Biosearch 9600). Alternatively,the follistatin polypeptides, fragments or variants thereof may berecombinantly produced using various expression systems (e.g., E. coli,Chinese Hamster Ovary cells, COS cells, baculovirus) as is well known inthe art (also see below). In a further embodiment, the modified orunmodified follistatin polypeptides may be produced by digestion ofnaturally occurring or recombinantly produced full-length follistatinpolypeptides by using, for example, a protease, e.g., trypsin,thermolysin, chymotrypsin, pepsin, or paired basic amino acid convertingenzyme (PACE). Computer analysis (using a commercially availablesoftware, e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.)can be used to identify proteolytic cleavage sites. Alternatively, suchfollistatin polypeptides may be produced from naturally occurring orrecombinantly produced full-length follistatin polypeptides such asstandard techniques known in the art, such as by chemical cleavage(e.g., cyanogen bromide, hydroxylamine).

3. Nucleic Acids Encoding Follistatin Polypeptides

In certain aspects, the invention provides isolated and/or recombinantnucleic acids encoding any of the follistatin polypeptides disclosedherein. The subject nucleic acids may be single-stranded or doublestranded. Such nucleic acids may be DNA or RNA molecules. These nucleicacids are may be used, for example, in methods for making follistatinpolypeptides.

For example, the following sequence encodes a naturally occurring humanfollistatin precursor polypeptide (SEQ ID NO: 19) (NCBI Accession NumberBC004107.2, 1032 bp):

atggtccgcgcgaggcaccagccgggtgggctttgcctcctgctgctgctgctctgccagttcatggaggaccgcagtgcccaggctgggaactgctggctccgtcaagcgaagaacggccgctgccaggtcctgtacaagaccgaactgagcaaggaggagtgctgcagcaccggccggctgagcacctcgtggaccgaggaggacgtgaatgacaacacactcttcaagtggatgattttcaacgggggcgcccccaactgcatcccctgtaaagaaacgtgtgagaacgtggactgtggacctgggaaaaaatgccgaatgaacaagaagaacaaaccccgctgcgtctgcgccccggattgttccaacatcacctggaagggtccagtctgcgggctggatgggaaaacctaccgcaatgaatgtgcactcctaaaggcaagatgtaaagagcagccagaactggaagtccagtaccaaggcagatgtaaaaagacttgtcgggatgttttctgtccaggcagctccacatgtgtggtggaccagaccaataatgcctactgtgtgacctgtaatcggatttgcccagagcctgcttcctctgagcaatatctctgtgggaatgatggagtcacctactccagtgcctgccacctgagaaaggctacctgcctgctgggcagatctattggattagcctatgagggaaagtgtatcaaagcaaagtcctgtgaagatatccagtgcactggtgggaaaaaatgtttatgggatttcaaggttgggagaggccggtgttccctctgtgatgagctgtgccctgacagtaagtcggatgagcctgtctgtgccagtgacaatgccacttatgccagcgagtgtgccatgaaggaagctgcctgctcctcaggtgtgctactggaagtaaagcactccggatcttgcaactccatttcggaagacaccgaggaagaggaggaagatgaagaccaggactacagctttcctatatcttctattctagagtgg

The following sequence encodes the mature FST(315) polypeptide (SEQ IDNO: 20).

gggaactgctggctccgtcaagcgaagaacggccgctgccaggtcctgtacaagaccgaactgagcaaggaggagtgctgcagcaccggccggctgagcacctcgtggaccgaggaggacgtgaatgacaacacactcttcaagtggatgattttcaacgggggcgcccccaactgcatcccctgtaaagaaacgtgtgagaacgtggactgtggacctgggaaaaaatgccgaatgaacaagaagaacaaaccccgctgcgtctgcgccccggattgttccaacatcacctggaagggtccagtctgcgggctggatgggaaaacctaccgcaatgaatgtgcactcctaaaggcaagatgtaaagagcagccagaactggaagtccagtaccaaggcagatgtaaaaagacttgtcgggatgttttctgtccaggcagctccacatgtgtggtggaccagaccaataatgcctactgtgtgacctgtaatcggatttgcccagagcctgcttcctctgagcaatatctctgtgggaatgatggagtcacctactccagtgcctgccacctgagaaaggctacctgcctgctgggcagatctattggattagcctatgagggaaagtgtatcaaagcaaagtcctgtgaagatatccagtgcactggtgggaaaaaatgtttatgggatttcaaggttgggagaggccggtgttccctctgtgatgagctgtgccctgacagtaagtcggatgagcctgtctgtgccagtgacaatgccacttatgccagcgagtgtgccatgaaggaagctgcctgctcctcaggtgtgctactggaagtaaagcactccggatcttgcaactccatttcggaagacaccgaggaagaggaggaagatgaagaccaggactacagctttcctatatcttctattctagagtgg

The following sequence encodes the FST(288) polypeptide (SEQ ID NO: 21).

gggaactgctggctccgtcaagcgaagaacggccgctgccaggtcctgtacaagaccgaactgagcaaggaggagtgctgcagcaccggccggctgagcacctcgtggaccgaggaggacgtgaatgacaacacactcttcaagtggatgattttcaacgggggcgcccccaactgcatcccctgtaaagaaacgtgtgagaacgtggactgtggacctgggaaaaaatgccgaatgaacaagaagaacaaaccccgctgcgtctgcgccccggattgttccaacatcacctggaagggtccagtctgcgggctggatgggaaaacctaccgcaatgaatgtgcactcctaaaggcaagatgtaaagagcagccagaactggaagtccagtaccaaggcagatgtaaaaagacttgtcgggatgttttctgtccaggcagctccacatgtgtggtggaccagaccaataatgcctactgtgtgacctgtaatcggatttgcccagagcctgcttcctctgagcaatatctctgtgggaatgatggagtcacctactccagtgcctgccacctgagaaaggctacctgcctgctgggcagatctattggattagcctatgagggaaagtgtatcaaagcaaagtcctgtgaagatatccagtgcactggtgggaaaaaatgtttatgggatttcaaggttgggagaggccggtgttccctctgtgatgagctgtgccctgacagtaagtcggatgagcctgtctgtgccagtgacaatgccacttatgccagcgagtgtgccatgaaggaagctgcctgctcctcaggtgtgctactggaagtaaagcact ccggatcttgcaac

The following sequence encodes the mature FST(291) polypeptide (SEQ IDNO: 22).

gggaactgctggctccgtcaagcgaagaacggccgctgccaggtcctgtacaagaccgaactgagcaaggaggagtgctgcagcaccggccggctgagcacctcgtggaccgaggaggacgtgaatgacaacacactcttcaagtggatgattttcaacgggggcgcccccaactgcatcccctgtaaagaaacgtgtgagaacgtggactgtggacctgggaaaaaatgccgaatgaacaagaagaacaaaccccgctgcgtctgcgccccggattgttccaacatcacctggaagggtccagtctgcgggctggatgggaaaacctaccgcaatgaatgtgcactcctaaaggcaagatgtaaagagcagccagaactggaagtccagtaccaaggcagatgtaaaaagacttgtcgggatgttttctgtccaggcagctccacatgtgtggtggaccagaccaataatgcctactgtgtgacctgtaatcggatttgcccagagcctgcttcctctgagcaatatctctgtgggaatgatggagtcacctactccagtgcctgccacctgagaaaggctacctgcctgctgggcagatctattggattagcctatgagggaaagtgtatcaaagcaaagtcctgtgaagatatccagtgcactggtgggaaaaaatgtttatgggatttcaaggttgggagaggccggtgttccctctgtgatgagctgtgccctgacagtaagtcggatgagcctgtctgtgccagtgacaatgccacttatgccagcgagtgtgccatgaaggaagctgcctgctcctcaggtgtgctactggaagtaaagcactccggatcttgcaactccatttcgtgg

In certain aspects, the subject nucleic acids encoding follistatinpolypeptides are further understood to include nucleic acids that arevariants of SEQ ID NOs: 19-22. Variant nucleotide sequences includesequences that differ by one or more nucleotide substitutions, additionsor deletions, such as allelic variants; and will, therefore, includecoding sequences that differ from the nucleotide sequence of the codingsequence designated in SEQ ID NOs: 19-22.

In certain embodiments, the disclosure provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 96% 97%,98%, 99%, or 100% identical to SEQ ID NOs: 19-22, and particularly thoseportions thereof that are derived from follistatin (nucleotidescorresponding to amino acids 95-164 of SEQ ID NO:1). One of ordinaryskill in the art will appreciate that nucleic acid sequencescomplementary to SEQ ID NOs: 19-22, and variants of SEQ ID NO: 19-22 arealso within the scope of this disclosure. In further embodiments, thenucleic acid sequences of the disclosure can be isolated, recombinant,and/or fused with a heterologous nucleotide sequence, or in a DNAlibrary.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequence designated in SEQ ID NOs: 19-22, complementsequence of SEQ ID NOs: 19-22, or fragments thereof (e.g., nucleotides19-22).

One of ordinary skill in the art will understand readily thatappropriate stringency conditions that promote DNA hybridization can bevaried. One of ordinary skill in the art will understand readily thatappropriate stringency conditions which promote DNA hybridization can bevaried. For example, one could perform the hybridization at 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In one embodiment, the invention provides nucleic acids which hybridizeunder low stringency conditions of 6×SSC at room temperature followed bya wash at 2×SSC at room temperature.

Isolated nucleic acids that differ from the nucleic acids as set forthin SEQ ID NOs: 19-22 due to degeneracy in the genetic code are alsowithin the scope of the disclosure. For example, a number of amino acidsare designated by more than one triplet. Codons that specify the sameamino acid, or synonyms (for example, CAU and CAC are synonyms forhistidine) may result in “silent” mutations that do not affect the aminoacid sequence of the protein. However, it is expected that DNA sequencepolymorphisms that do lead to changes in the amino acid sequences of thesubject proteins will exist among mammalian cells. One skilled in theart will appreciate that these variations in one or more nucleotides (upto about 3-5% of the nucleotides) of the nucleic acids encoding aparticular protein may exist among individuals of a given species due tonatural allelic variation. Any and all such nucleotide variations andresulting amino acid polymorphisms are within the scope of thisdisclosure.

In certain embodiments, the recombinant nucleic acids of the disclosuremay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the disclosure. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects, the subject nucleic acid is provided in anexpression vector comprising a nucleotide sequence encoding afollistatin polypeptide and operably linked to at least one regulatorysequence. Regulatory sequences are art-recognized and are selected todirect expression of the follistatin polypeptide. Accordingly, the termregulatory sequence includes promoters, enhancers, and other expressioncontrol elements. Exemplary regulatory sequences are described inGoeddel; Gene Expression Technology: Methods in Enzymology, AcademicPress, San Diego, Calif. (1990). For instance, any of a wide variety ofexpression control sequences that control the expression of a DNAsequence when operatively linked to it may be used in these vectors toexpress DNA sequences encoding a follistatin polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid of the disclosure can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant follistatin polypeptide include plasmids and other vectors.For instance, suitable vectors include plasmids of the types:pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids,pBTac-derived plasmids and pUC-derived plasmids for expression inprokaryotic cells, such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 1989) Chapters 16 and 17. In some instances, it may bedesirable to express the recombinant polypeptides by the use of abaculovirus expression system. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derivedvectors (such as the ß-gal containing pBlueBac III).

In certain embodiments, a vector will be designed for production of thesubject follistatin polypeptides in CHO cells, such as a Pcmv-Scriptvector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen,Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As willbe apparent, the subject gene constructs can be used to cause expressionof the subject follistatin polypeptides in cells propagated in culture,e.g., to produce proteins, including fusion proteins or variantproteins, for purification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NOs: 19-22)for one or more of the subject follistatin polypeptides. The host cellmay be any prokaryotic or eukaryotic cell. For example, a follistatinpolypeptide of the disclosure may be expressed in bacterial cells suchas E. coli, insect cells (e.g., using a baculovirus expression system),yeast, or mammalian cells. Other suitable host cells are known to thoseskilled in the art.

Accordingly, the present disclosure further pertains to methods ofproducing the subject follistatin polypeptides. For example, a host celltransfected with an expression vector encoding a follistatin polypeptidecan be cultured under appropriate conditions to allow expression of thefollistatin polypeptide to occur. The follistatin polypeptide may besecreted and isolated from a mixture of cells and medium containing thefollistatin polypeptide. Alternatively, the follistatin polypeptide maybe retained cytoplasmically or in a membrane fraction and the cellsharvested, lysed and the protein isolated. A cell culture includes hostcells, media and other byproducts. Suitable media for cell culture arewell known in the art. The subject follistatin polypeptides can beisolated from cell culture medium, host cells, or both, using techniquesknown in the art for purifying proteins, including ion-exchangechromatography, gel filtration chromatography, ultrafiltration,electrophoresis, and immunoaffinity purification with antibodiesspecific for particular epitopes of the follistatin polypeptides. In apreferred embodiment, the follistatin polypeptide is a fusion proteincontaining a domain that facilitates its purification.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant follistatinpolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified follistatin polypeptide (e.g., seeHochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al.,PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers that give rise tocomplementary overhangs between two consecutive gene fragments that cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

4. Exemplary Therapeutic Uses

In certain embodiments, compositions of the present disclosure,including for example FST(288)-IgG1, FST(288)-IgG2, FST(291)-IgG1,FST(291)-IgG2, FST(315)-IgG1, FST(315)-IgG2, and any of the otherfollistatin polypeptides disclosed herein, can be used for treating orpreventing a disease or condition that is described in this section,including diseases or disorders that are associated with abnormalactivity of a follistatin polypeptide and/or a follistatin ligand (e.g.,GDF8). These diseases, disorders or conditions are generally referred toherein as “follistatin-associated conditions.” In certain embodiments,the present disclosure provides methods of treating or preventing anindividual in need thereof through administering to the individual atherapeutically effective amount of a follistatin polypeptide asdescribed above. These methods are particularly aimed at therapeutic andprophylactic treatments of animals, and more particularly, humans.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes amelioration or elimination of the condition once it has beenestablished.

Follistatin-ligand complexes play essential roles in tissue growth aswell as early developmental processes such as the correct formation ofvarious structures or in one or more post-developmental capacitiesincluding sexual development, pituitary hormone production, and creationof muscle. Thus, follistatin-associated conditions include abnormaltissue growth and developmental defects.

Exemplary conditions for treatment include neuromuscular disorders(e.g., muscular dystrophy and muscle atrophy), congestive obstructivepulmonary disease (and muscle wasting associated with COPD), musclewasting syndrome, sarcopenia, and cachexia. Other exemplary conditionsinclude musculodegenerative and neuromuscular disorders, tissue repair(e.g., wound healing), and neurodegenerative diseases (e.g., amyotrophiclateral sclerosis).

In certain embodiments, compositions (e.g., FST-Fc polypeptides) of theinvention are used as part of a treatment for a muscular dystrophy. Theterm “muscular dystrophy” refers to a group of degenerative musclediseases characterized by gradual weakening and deterioration ofskeletal muscles and sometimes the heart and respiratory muscles.Muscular dystrophies are genetic disorders characterized by progressivemuscle wasting and weakness that begin with microscopic changes in themuscle. As muscles degenerate over time, the person's muscle strengthdeclines. Exemplary muscular dystrophies that can be treated with aregimen including the subject follistatin polypeptides include: DuchenneMuscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD),Emery-Dreifuss Muscular Dystrophy (EDMD), Limb-Girdle Muscular Dystrophy(LGMD), Facioscapulohumeral Muscular Dystrophy (FSH or FSHD) (also knownas Landouzy-Dejerine), Myotonic Dystrophy (MMD) (also known asSteinert's Disease), Oculopharyngeal Muscular Dystrophy (OPMD), DistalMuscular Dystrophy (DD), Congenital Muscular Dystrophy (CMD).

Duchenne Muscular Dystrophy (DMD) was first described by the Frenchneurologist Guillaume Benjamin Amand Duchenne in the 1860s. BeckerMuscular Dystrophy (BMD) is named after the German doctor Peter EmilBecker, who first described this variant of DMD in the 1950s. DMD is oneof the most frequent inherited diseases in males, affecting one in 3,500boys. DMD occurs when the dystrophin gene, located on the short arm ofthe X chromosome, is broken. Since males only carry one copy of the Xchromosome, they only have one copy of the dystrophin gene. Without thedystrophin protein, muscle is easily damaged during cycles ofcontraction and relaxation. While early in the disease musclecompensates by regeneration, later on muscle progenitor cells cannotkeep up with the ongoing damage and healthy muscle is replaced bynon-functional fibro-fatty tissue.

BMD results from different mutations in the dystrophin gene. BMDpatients have some dystrophin, but it is either insufficient in quantityor poor in quality. Having some dystrophin protects the muscles of thosewith BMD from degenerating as badly or as quickly as those of peoplewith DMD.

For example, recent research demonstrates that blocking or eliminatingfunction of GDF8 (a follistatin ligand) in vivo can effectively treat atleast certain symptoms in DMD and BMD patients. Thus, the subjectfollistatin polypeptides may act as GDF8 inhibitors (antagonists), andconstitute an alternative means of blocking the functions of GDF8 invivo in DMD and BMD patients.

Similarly, the subject follistatin polypeptides provide an effectivemeans to increase muscle mass in other disease conditions that are inneed of muscle growth. For example, ALS, also called Lou Gehrig'sdisease (motor neuron disease) is a chronic, incurable, and unstoppableCNS disorder that attacks the motor neurons, components of the CNS thatconnect the brain to the skeletal muscles. In ALS, the motor neuronsdeteriorate and eventually die, and though a person's brain normallyremains fully functioning and alert, the command to move never reachesthe muscles. Most people who get ALS are between 40 and 70 years old.The first motor neurons that weaken are those leading to the arms orlegs. Those with ALS may have trouble walking, they may drop things,fall, slur their speech, and laugh or cry uncontrollably. Eventually themuscles in the limbs begin to atrophy from disuse. This muscle weaknesswill become debilitating and a person will need a wheel chair or becomeunable to function out of bed. Most ALS patients die from respiratoryfailure or from complications of ventilator assistance like pneumonia,3-5 years from disease onset.

Charcot-Marie-Tooth Disease (CMT) may be treated by local administrationof the follistatin polypeptides described herein. CMT is a group ofinherited disorders affecting the peripheral nerves and resulting inprogressive, and often local, muscle weakness and degeneration. Aspectsof the disease that may be treated include foot deformity (very higharched feet); foot drop (inability to hold foot horizontal); “Slapping”gait (feet slap on the floor when walking because of foot drop); loss ofmuscle in the lower legs; numbness in the feet; difficulty with balance;or weakness in the arms and hands.

Muscles of patients with a variety of systemic muscle disorders may betreated with the follistatin polypeptides disclosed herein, including:Lambert-Eaton Myasthenic Syndrome (LEMS); Metabolic Dystrophies; SpinalMuscular Atrophy (SMA); Dermatomyositis (DM); Distal Muscular Dystrophy(DD); Emery-Dreifuss Muscular Dystrophy (EDMD); Endocrine Myopathies;Friedreich's Ataxia (FA); Inherited Myopathies; Mitochondrial Myopathy;Myasthenia Gravis (MG); Polymyositis (PM).

Muscles of patients with a post-surgical or disuse atrophy of one ormuscles may be treated with the follistatin polypeptides disclosedherein including atrophy after: Hip Fracture; Total Hip Arthroplasty(THA); Total Knee Arthroplasty (TKA) or Rotator Cuff surgery.

Muscles of patients suffering from a variety of other diseases thatcause muscle loss or weakening may be treated with the follistatinpolypeptides disclosed herein, including muscles of patients with thefollowing diseases: sarcopenia, cachexia, various types of cancer,including lung, colon and ovarian cancer, patients on long termventilation assistance, diabetes, chronic obstructive pulmonarydisorder, renal failure, cardiac failure, trauma and disorders of theperipheral nerves.

Follistatin polypeptide-induced increased muscle mass might also benefitthose suffering from muscle wasting diseases. GDF8 expression correlatesinversely with fat-free mass in humans and that increased expression ofthe GDF8 gene is associated with weight loss in men with AIDS wastingsyndrome. By inhibiting the function of GDF8 in AIDS patients, at leastcertain symptoms of AIDS may be alleviated, if not completelyeliminated, thus significantly improving quality of life in AIDSpatients.

5. Pharmaceutical Compositions

In certain embodiments, compounds (e.g., follistatin polypeptides) ofthe present invention are formulated with a pharmaceutically acceptablecarrier. For example, a follistatin polypeptide can be administeredalone or as a component of a pharmaceutical formulation (i.e., atherapeutic composition). The subject compounds may be formulated foradministration in any convenient way for use in human or veterinarymedicine.

In certain embodiments, the therapeutic method of the invention includesadministering the composition topically, systemically, or locally as animplant or device. When administered, the therapeutic composition foruse in this invention is, of course, in a pyrogen-free, physiologicallyacceptable form. Further, the composition may desirably be encapsulatedor injected in a viscous form for delivery to a target tissue site(e.g., bone, cartilage, muscle, fat or neurons), for example, a sitehaving tissue damage. Topical administration may be suitable for woundhealing and tissue repair. Therapeutically useful agents other than thefollistatin polypeptides, which may also optionally be included in thecomposition as described above, may alternatively or additionally, beadministered simultaneously or sequentially with the subject compounds(e.g., follistatin polypeptides) in the methods of the invention.

In certain embodiments, compositions of the present invention mayinclude a matrix capable of delivering one or more therapeutic compounds(e.g., follistatin polypeptides) to a target tissue site, providing astructure for the developing tissue and optimally capable of beingresorbed into the body. For example, the matrix may provide slow releaseof the follistatin polypeptides. Such matrices may be formed ofmaterials presently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above-mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as cetyl alcohol andglycerol monostearate; (8) absorbents, such as kaolin and bentoniteclay; (9) lubricants, such a talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof;and (10) coloring agents. In the case of capsules, tablets and pills,the pharmaceutical compositions may also comprise buffering agents.Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugars, as well as high molecular weight polyethylene glycols.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Certain compositions disclosed herein may be administered topically,either to skin or to mucosal membranes. The topical formulations mayfurther include one or more of the wide variety of agents known to beeffective as skin or stratum corneum penetration enhancers. Examples ofthese are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide,dimethylformamide, propylene glycol, methyl or isopropyl alcohol,dimethyl sulfoxide, and azone. Additional agents may further be includedto make the formulation cosmetically acceptable. Examples of these arefats, waxes, oils, dyes, fragrances, preservatives, stabilizers, andsurface active agents. Keratolytic agents such as those known in the artmay also be included. Examples are salicylic acid and sulfur.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that may be required. Theointments, pastes, creams and gels may contain, in addition to a subjectcompound of the invention (e.g., a follistatin polypeptide), excipients,such as animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a subject compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

In certain embodiments, pharmaceutical compositions suitable forparenteral administration may comprise one or more follistatinpolypeptides in combination with one or more pharmaceutically acceptablesterile isotonic aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, or sterile powders which may be reconstitutedinto sterile injectable solutions or dispersions just prior to use,which may contain antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers that may be employed in the pharmaceuticalcompositions of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician, considering various factors that modify the actionof the subject compounds of the invention (e.g., follistatinpolypeptides). The various factors will depend upon the disease to betreated.

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of follistatin polypeptides or othercompounds disclosed herein. Such therapy would achieve its therapeuticeffect by introduction of the follistatin polynucleotide sequences intocells or tissues having the disorders as listed above. Delivery offollistatin polynucleotide sequences can be achieved using a recombinantexpression vector such as a chimeric virus or a colloidal dispersionsystem. Preferred for therapeutic delivery of follistatin polynucleotidesequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. Retroviral vectors can be madetarget-specific by attaching, for example, a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody. Thoseof skill in the art will recognize that specific polynucleotidesequences can be inserted into the retroviral genome or attached to aviral envelope to allow target specific delivery of the retroviralvector containing the follistatin polynucleotide. In one preferredembodiment, the vector is targeted to bone, cartilage, muscle or neuroncells/tissues.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for follistatin polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

Exemplification

The invention now being generally described will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustrating certain embodiments of the presentinvention. These examples are not intended to limit the invention.

Example 1: Generation of Follistatin-Fc Proteins

Follistatin (FST) is known to have complex pharmacokinetic behavior. Theshort form FST(288) is reported to be more effective at blocking ligandsand binds to cell surfaces in part due to its unmasked heparin bindingdomain. FST(315) is thought to be less effective but less attracted tocell surfaces due to the acid rich C-terminal amino acid sequence, whichneutralizes the heparin binding domain. In the literature, follistatinis generally reported as having systemic effects. Applicants sought todetermine whether a follistatin construct could be designed that wouldtend to have effects in the tissue of administration (such as aninjected muscle), and whether dimerization of follistatin would provideenhanced tissue retention. The Fc domains of immunoglobulins are knownto form dimers. To explore the effects of follistatin-Fc fusion proteinson muscle and other tissues, and to evaluate the effects of Fc-mediateddimerization on the pharmacokinetic properties of follistatinpolypeptides, Applicants generated fusion proteins containing FST(288)or FST(315) fused to an Fc portion of an IgG1. A TGGG linker sequencewas selected to join each follistatin polypeptide to the Fc portion.

For each FST-IgG1 construct, the following three leader sequences wereconsidered:

(1) Follistatin leader: (SEQ ID NO: 23) MVRARHQPGGLCLLLLLLCQFMEDRSAQA(2) Tissue plasminogen activator (TPA): (SEQ ID NO: 24)MDAMKRGLCCVLLLCGAVFVSP (3) Honey bee melittin (HBML): (SEQ ID NO: 25)MKFLVNVALVFMVVYISYIYA

The selected FST-Fc proteins incorporate the follistatin leader. TheFST(288)-IgG1 fusion has the unprocessed and mature amino acid sequencesshown below.

Unprocessed FST(288)-IgG1 (SEQ ID NO: 26)MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Mature FST(288)-IgG1 (SEQ ID NO: 27)GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK

The initial “GN” sequence may be removed, yielding the followingpolypeptide. (SEQ ID NO: 28)

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK

The FST(315)-IgG1 fusion has the unprocessed and mature amino acidsequences shown below.

Unprocessed FST(315)-IgG1 (SEQ ID NO: 29)MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEWTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Mature FST(315)-IgG1 (SEQ ID NO: 30)GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEWTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The initial “GN” sequence may be removed, yielding the followingpolypeptide. (SEQ ID NO: 31)

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEWTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Proteins were expressed in HEK-293 cells or CHO cells and purified fromconditioned media by filtration and protein A chromatography. In someinstances anion exchange and hydrophobic interaction chromatographyand/or gel filtration was also used.

Protein activity was assessed by binding to activin A or GDF11. In eachcase, the proteins bind with a K_(D) of less than 10 pM.

Example 2: The Effect of Systemic Administration of Follistatin-FcProteins on Muscle Mass and Strength in Mice

Applicants determined the ability of follistatin-Fc proteins to increasemuscle mass and strength in wild-type mice after systemicadministration. An ActRIIB-Fc fusion protein that is well-known tostimulate substantial whole-body increases in lean muscle mass was usedas a positive control.

C57BL/6 mice were dosed (10 mg/kg; subcutaneously (s.c.)) twice/week forfour weeks with the FST(288)-IgG1 protein, the human FST(315)-IgG1protein, or the human ActRIIB-Fc protein. Mice were subjected towhole-body nuclear magnetic resonance (NMR) scanning to determine thepercent change of whole body lean tissue mass. ActRIIB-Fc treated miceexhibited a significant (approximately 35%) increase in lean tissue whencompared to the vehicle-control group. Mice treated with either theFST(288)-IgG1 or FST(315)-IgG1 protein exhibited little increase in leantissue mass compared to the control cohort. See FIG. 2. At the end ofthe study, pectoralis, tibialis anterior (TA), gastrocnemius, andfemoris muscles were dissected and weighed. As shown in FIG. 4,ActRIIB-Fc treatment significantly increased muscle mass in each ofthese muscle groups. In contrast, little to no increase in muscle masswas observed in either the FST(288)-IgG1 or FST(315)-IgG1 treatmentgroups. See FIG. 2.

During the course of this study, mice were also examined for changes inmuscle strength. The force a mouse exerts when pulling a forcetransducer is measured to determine forelimb grip strength. Applicantsobserved that mice treated with the ActRIIB-Fc protein exhibitedincreased muscle strength. In contrast, there was no increase in gripstrength observed in either the FST(288)-IgG1 or FST(315)-IgG1 treatmentgroups. See FIG. 3.

Together, the results confirm that systemic administration of ActRIIB-Fcprofoundly increases both muscle mass and strength in mice when comparedto vehicle-control animals. In contrast, there was little to no increasein muscle mass or strength observed in mice treated with either thefollistatin-Fc fusion protein FST(288)-IgG1 or FST(315)-IgG1. Therefore,it appears that follistatin-Fc fusions proteins have little or no effecton muscle mass or strength in vivo when administered systemically.

Example 3: The Effect of Systemic Administration of Follistatin-FcProteins on FSH Levels

Follistatin is primarily characterized for its ability to bind andinhibit members of the TGF-beta superfamily of signaling proteins. Inparticular, follistatin is known to be a potent inhibitor of activinactivity. Activin is a potent inducer of follicle-stimulating hormone(FSH) production. FSH is synthesized and secreted by gonadotrophs of theanterior pituitary gland and regulates growth and development duringpubertal maturation and various reproductive processes in the body. Toassess systemic effects of follistatin-Fc polypeptides, effects on FSHlevels were evaluated.

Treatment (10 mg/kg; subcutaneously (s.c.) twice/week) withFST(288)-IgG1 resulted in circulating levels of the drug at 3.836(±5.22) μg/mL. Similar treatment with FST(315)-IgG1 resulted insubstantially higher serum levels of the drug at 19.31 (±1.85) μg/mL. Asindicated in FIG. 5, FST(288)-IgG1 did not have any significant effectson serum levels of FSH, suggesting that this FST(288)-IgG1 treatmentregime does not significantly affect systemic activin activity. Incontrast, FST(315)-IgG1 treatment resulted in a decrease in circulatinglevels of FSH, indicating that systemic administration of FST(315)-IgG1has an effect on systemic activin signaling. Overall, these dataindicate that use of a follistatin polypeptide with an unmasked heparinbinding domain, fused to an Fc domain that mediates dimerization, suchas FST(288)-IgG1 results in a protein that has little or no systemicactivity, while an FST(315)-IgG1, with a masked heparin binding domain,may be used to achieve systemic effects.

Example 4: The Effect of Local Administration of Follistatin-Fc Proteinson Muscle Mass and Strength in Mice

While there were no significant effects after systemic administration,Applicants used a similar experimental approach to determine iffollistatin can be used to locally increase muscle mass and strength inwild-type mice after intramuscular (i.m.) administration.

C57BL/6 mice were dosed (50 micrograms; i.m. into the rightgastrocnemius muscle) twice/week for four weeks with the FST(288)-Fcprotein, the FST(315)-Fc protein, or the human ActRIIB-Fc protein. Atvarious time points after initial treatment, mice were subjected towhole-body nuclear magnetic resonance (NMR) scanning to determine thepercent change of whole body lean tissue mass. ActRIIB-Fc treated miceexhibited a significant increase in lean tissue when compared to thevehicle-control group. In contrast, neither mice treated with theFST(288)-Fc nor FST(315)-Fc protein exhibited a significant increase inlean tissue mass compared to the control cohort. At the end of thestudy, both the right, injected gastrocnemius muscle and the left,contralateral gastrocnemius muscle were dissected and weighed. As shownin FIG. 6, ActRIIB-Fc treatment significantly increased muscle mass inboth the right and left gastrocnemius muscles in comparison tovehicle-treated mice. Therefore, ActRIIB-Fc has systemic effects onincreasing muscle mass even when restricted to local administration in asingle muscle. In contrast, both FST(288)-Fc and FST(315)-Fc resulted insignificant increases in muscle mass of the right gastrocnemius musclebut had no effect on the mass of the contralateral muscle. Therefore,contrary the effects observed after systemic administration, it appearsthat follistatin protein is a potent stimulator of muscle mass whendirectly administered into a muscle. Furthermore, follistatin appears tohave a distinct advantage over other agents like ActRIIB-Fc in that itseffects on muscle mass are localized to the site of administration,indicating that follistatin can be used for targeted therapy of aselected muscle, or muscle groups, without affecting the normalgrowth/activity of surrounding, non-targeted muscles.

Applicants also closely monitored the serum levels of follistatin-Fcfusion protein after i.m. administration. Treatment with FST(288)-IgG1resulted in a circulating levels of the drug at 0.156 (±0.245) m/mL.Similar treatment with FST(315)-IgG1 resulted in slightly higher serumlevels of the drug at 3.58 (±1.73) m/mL, but these levels weresubstantially lower than those observed after systemic administration ofFST(315)-IgG1. As both FST(288)-IgG1 and FST(315)-IgG1 circulate inpatient serum at lower levels after i.m. injection than is observedafter systemic administration of FST(288)-IgG1 (i.e., 3.836 (±5.22)m/mL), neither FST(288)-IgG1 nor FST(315)-IgG1 would be expected to havesignificant effects on serum levels of FSH as FST(288)-IgG1 had no sucheffect after s.c. administration. See FIG. 5. Accordingly, these dataindicate that both FST(288)-IgG1 and FST(315)-IgG1 would be particularlywell-suited for promoting targeted muscle growth in patients that arereproductively active or have a desire to minimize effects on thereproductive system.

A similar experiment was conducted to establish a dose-response curve ofthe effects of FST(288)-IgG1 on muscle mass and quality. C57BL/6 micewere dosed with varying amounts (1 to 100 micrograms); i.m. into theright gastrocnemius muscle twice/week for four weeks. As shown in FIG.8, the selective increase in the muscle mass of the injected muscleversus the contralateral muscle was greater with greater doses ofFST(288)-IgG1. Muscle cross sections revealed the enhanced muscle massto be the result of muscle fiber hypertrophy, rather than hypoplasia.

Example 5: Fc Optimization of Locally-Acting Follistatin-Fc FusionProteins

As described in the preceding Examples, follistatin-Fc fusion proteinssuch as FST(288)-IgG1 and FST(315)-IgG1 have poor systemic effects onmuscle and other tissues, and particularly FST(288) forms of the proteinare active at the site of injection. Applicants and others establishedthat FST(288) binds to cells by virtue of the heparin binding domain andthis binding can be eliminated by exogenous heparin. As a consequence,Applicants determined that immunoglobulin domains known to mediate CDCand ADCC effects on targeted cells may cause damage to cells treatedwith the heparin-binding follistatin constructs. Such damage couldmanifest as an immune reaction in the targeted tissue or in decreasedgrowth of the targeted tissue. Therefore Applicants generated versionsof follistatin polypeptides employing the Fc portion of human IgG2,which is an example of an IgG constant domain that is known to havediminished capability to stimulate CDC and ADCC activity. Thisexperiment was conducted to ascertain whether follistatin-Fc fusionproteins using alternative Fc domains would retain activity.

Applicants generated fusion proteins containing FST(288) or FST(315)fused to an Fc portion of an IgG2. A TGGG linker sequence was selectedto join each follistatin polypeptide to the Fc portion.

For each FST-IgG2 construct, the follistatin leader was employed.

The FST(288)-IgG2 fusion has the unprocessed and mature amino acidsequences shown below.

Unprocessed FST(288)-IgG2 (SEQ ID NO: 32)MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Which is encoded by the following nucleic acid sequence (SEQ ID NO:44)

atggtccgcgcgaggcaccagccgggtgggctttgcctcctgctgctgctgctctgccagttcatggaggaccgcagtgcccaggctgggaactgctggctccgtcaagcgaagaacggccgctgccaggtcctgtacaagaccgaactgagcaaggaggagtgctgcagcaccggccggctgagcacctcgtggaccgaggaggacgtgaatgacaacacactcttcaagtggatgattttcaacgggggcgcccccaactgcatcccctgtaaagaaacgtgtgagaacgtggactgtggacctgggaaaaaatgccgaatgaacaagaagaacaaaccccgctgcgtctgcgccccggattgttccaacatcacctggaagggtccagtctgcgggctggatgggaaaacctaccgcaatgaatgtgcactcctaaaggcaagatgtaaagagcagccagaactggaagtccagtaccaaggcagatgtaaaaagacttgtcgggatgttttctgtccaggcagctccacatgtgtggtggaccagaccaataatgcctactgtgtgacctgtaatcggatttgcccagagcctgcttcctctgagcaatatctctgtgggaatgatggagtcacctactccagtgcctgccacctgagaaaggctacctgcctgctgggcagatctattggattagcctatgagggaaagtgtatcaaagcaaagtcctgtgaagatatccagtgcactggtgggaaaaaatgtttatgggatttcaaggttgggagaggccggtgttccctctgtgatgagctgtgccctgacagtaagtcggatgagcctgtctgtgccagtgacaatgccacttatgccagcgagtgtgccatgaaggaagctgcctgctcctcaggtgtgctactggaagtaaagcactccggatcttgcaacaccggtggtggagtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgtcgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagaattc

Mature FST(288)-IgG2 (SEQ ID NO: 33)GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK

The initial “GN” sequence may be removed, yielding the followingpolypeptide. (SEQ ID NO: 34)

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK

The FST(315)-IgG2 fusion has the unprocessed and mature amino acidsequences shown below.

Unprocessed FST(315)-IgG2 (SEQ ID NO: 35)MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEWTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK

Which is encoded by the following nucleic acid sequence (SEQ ID NO:45)

atggtccgcgcgaggcaccagccgggtgggctttgcctcctgctgctgctgctctgccagttcatggaggaccgcagtgcccaggctgggaactgctggctccgtcaagcgaagaacggccgctgccaggtcctgtacaagaccgaactgagcaaggaggagtgctgcagcaccggccggctgagcacctcgtggaccgaggaggacgtgaatgacaacacactcttcaagtggatgattttcaacgggggtgcccccaactgcatcccctgtaaagaaacgtgtgagaacgtggactgtggacctgggaaaaaatgccgaatgaacaagaagaacaaaccccgctgcgtctgcgccccggattgttccaacatcacctggaagggtccagtctgcgggctggatgggaaaacctaccgcaatgaatgtgcactcctaaaggcaagatgtaaagagcagccagaactggaagtccagtaccaaggcagatgtaaaaagacttgtcgggatgttttctgtccaggcagctccacatgtgtggtggaccagaccaataatgcctactgtgtgacctgtaatcggatttgcccagagcctgcttcctctgagcaatatctctgtgggaatgatggagtcacctactccagtgcctgccacctgagaaaggctacctgcctgctgggcagatctattggattagcctatgagggaaagtgtatcaaagcaaagtcctgtgaagatatccagtgcactggtgggaaaaaatgtttatgggatttcaaggttgggagaggccggtgttccctctgtgatgagctgtgccctgacagtaagtcggatgagcctgtctgtgccagtgacaatgccacttatgccagcgagtgtgccatgaaggaagctgcctgctcctcaggtgtgctactggaagtaaagcactccggatcttgcaactccatttcggaagacaccgaggaagaggaggaagatgaagaccaggactacagctttcctatatcttctattctagagtggaccggtggtggagtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgtcgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagaattc

Mature FST(315)-IgG2 (SEQ ID NO: 36)GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEWTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The initial “GN” sequence may be removed, yielding the followingpolypeptide. (SEQ ID NO: 37)

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEWTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKIKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Proteins were expressed in HEK-293 cells or CHO cells and purified fromconditioned media by filtration and protein A chromatography. In someinstances anion exchange and hydrophobic interaction chromatographyand/or gel filtration was also used.

Protein activity was assessed by binding to activin A or GDF11. In eachcase, the proteins bind with a K_(D) of less than 10 pM. These dataindicate that Follistatin-IgG2 fusion proteins can be generated andexpressed and retain picomolar ligand binding activity.

Example 6: Optimized Locally-Acting Follistatin-Fc Fusion Proteins

To assess whether an optimal follistatin-Fc fusion protein could begenerated, a variety of truncations between the C-terminus of FST(288)and FST(315) were generated. One of these truncations, ending at aminoacid 291 and termed FST(291) showed superior expression propertiescompared to other forms and retained the desired heparin bindingactivity, despite containing a small portion of the masking domain ofFST(315). This form was fused to the Fc portion of human IgG1 and IgG2to generate FST(291)-IgG1 and FST(291)-IgG2.

A TGGG linker sequence was selected to join each follistatin polypeptideto the Fc portion.

For each FST-IgG1 construct, the follistatin leader was employed.

The FST(291)-IgG1 fusion has the unprocessed and mature amino acidsequences shown below.

Unprocessed FST(291)-IgG1 (SEQ ID NO: 38)MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Mature FST(291)-IgG1 (SEQ ID NO: 39)GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK

The initial “GN” sequence may be removed, yielding the followingpolypeptide. (SEQ ID NO: 40)

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK

The FST(291)-IgG2 fusion has the unprocessed and mature amino acidsequences shown below.

Unprocessed FST(291)-IgG2 (SEQ ID NO: 41)MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Mature FST(291)-IgG2 (SEQ ID NO: 42)GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK

The initial “GN” sequence may be removed, yielding the followingpolypeptide. (SEQ ID NO: 43)

CWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISTGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK

Proteins were expressed in HEK-293 cells or CHO cells and purified fromconditioned media by filtration and protein A chromatography. In someinstances anion exchange and hydrophobic interaction chromatographyand/or gel filtration was also used.

Protein activity was assessed by binding to activin A or GDF11. In eachcase, the proteins bind with a K_(D) of less than 10 pM.

Additional truncation experiments were conducted to identifyFollistatin-IgG2 constructs, in the context of the TGGG linker,exhibiting an optimal ligand and heparin binding activity, so as togenerate a polypeptide with high potency, a strong tendency towardsretention in the treated tissue and low tendency to produce inflammatoryor immune reaction in the treated tissue. For this purpose a series ofconstructs were generated, termed FST(278)-IgG2, FST(284)-IgG2,FST(291)-IgG2 and FST(303)-IgG2 and compared to each other and toFST(288)-IgG2 and FST-(315)-IgG2. Heparin binding was assessed bymeasuring protein recovery from cells in the presence or absence ofheparin, quantitated by ELISA and expressed as a ratio of proteinrecovered in the presence of heparin to the protein recovered in theabsence of heparin. As shown in the table below, FST(278)-IgG2,FST(284)-IgG2, FST(288)-IgG2 and FST(291)-IgG2 all show similar ratiosof 3.00-4.00, while FST(303)-IgG2 and FST(315)-IgG2 show ratios of 1.50and 0.97, respectively. This indicates that as more amino acids areincluded between position 291 and 303, the heparin binding activity issharply reduced.

Heparin Binding of FST-IgG2 Truncations

Ratio (protein recovered with heparin/protein recovered FST-IgG2Construct without heparin) FST(278)-IgG2 4.18 FST(284)-IgG2 3.54FST(288)-IgG2 3.34 FST(291)-IgG2 3.00 FST(303)-IgG2 1.50 FST(315)-IgG20.97

Cell-based reporter gene assays (A-204 Reporter Gene Assay, described inWO/2006/012627) to assess inhibition of activin and GDF11 wereconducted. As shown in the table below, constructs extending beyondposition 288 provided enhanced ligand inhibition.

Ligand Inhibition of FST-IgG2 Truncations

IC50 (ng/ml) IC50 (ng/ml) FST-IgG2 Construct Activin A GDF-11FST(278)-IgG2 521 91 FST(284)-IgG2 369 123 FST(288)-IgG2 30 41FST(291)-IgG2 20 26 FST(303)-IgG2 2 18 FST(315)-IgG2 10 15

Taking together the heparin binding and ligand inhibition data, it isapparent that FST-IgG2 constructs, in the context of the TGGG linkerused here, or similar sized linkers (e.g., linkers sized 1-10 aminoacids, optionally 3-8 amino acids), that end at position 291-302 willhave enhanced ligand inhibition relative to FST(288)-IgG2 and enhancedheparin binding relative to FST(315)-IgG2, and that FST(291)-IgG2represents an optimal protein for local administration and effect.

Example 7: The Effect of Local Administration of FST(291)-IgG2 Proteinon Muscle Mass and Strength in Mice

Applicants assessed the activity of the optimized FST(291)-IgG2 proteinas used to locally increase muscle mass and strength in wild-type miceafter intramuscular (i.m.) administration.

C57BL/6 mice were dosed (100 micrograms in 50 microliters; i.m. into theleft gastrocnemius muscle) twice/week for four weeks with vehicle (PBS),FST(291)-IgG2 or a control Fc from IgG1. At the end of the study, boththe left, injected gastrocnemius muscle and the right, contralateralgastrocnemius muscle were dissected and weighed. As shown in FIG. 9,FST(291)-IgG2 treatment significantly increased muscle mass in theinjected left gastrocnemius muscles, to a remarkable degree, incomparison to vehicle-treated mice, with no effect observed on thecontralateral muscle. Additionally, pectoral and femoris muscles wereweighed and showed no change as a consequence of vehicle orFST(291)-IgG2 administration. Therefore, FST(291)-IgG2 has restrictedeffect on the injected muscle group with little or no systemic effect.Similar experiments have been conducted but injecting different musclegroups, including the triceps and the tibialis anterior. In each case,selective hypertrophy of the injected muscle was observed.

Additional experiments were conducted to directly compare the effects ofFST(288)-IgG1 and FST(291)-IgG2 on muscle growth. While both constructspromoted significant increased muscle mass in the injected muscle(gastrocnemius), the FST(291)-IgG2 caused approximately a 42% increasein the injected muscle versus the contralateral muscle, whileFST(288)-IgG1 caused approximately a 22% increase in injected muscleversus the contralateral muscle.

Accordingly, these data indicate that FST(291)-IgG2 is an optimalcompound for promoting targeted muscle growth in patients in needthereof.

Example 8: The Effect of Local Administration of FST(291)-IgG2 Proteinon Muscle in a Mouse Model of Duchenne Muscular Dystrophy

The effect of FST(291)-IgG2 on muscle mass was assessed in a mouse modelof Duchenne muscular dystrophy. The C57BL/10ScCN-Dmd^(mdx)/J (mdx)strain of mice is a well-established model of human Duchenne musculardystrophy (Bulfield, Siller et al. 1984; Partridge 2013).

Two separate studies were performed with mdx mice and the wild-typebackground strain, C57BL/10SnJ (WT). In the first study, treatment(either FST(291)-IgG2 or vehicle control) was initiated when micereached 6 weeks of age. In the second study, treatment was initiatedwhen mice reached 4 weeks of age. In both studies, mice received 100 μgFST(291)-IgG2 intramuscularly into the left gastrocnemius muscle, twiceper week, in a fixed volume of 50 μL per injection. Four-week old micewere treated for 4 weeks, and 6-week old mice were treated for 6 weeks.

At necropsy the gastrocnemius muscles from the injected (left) andcontralateral, non-injected (right) leg were excised and weighed. Inboth studies the injected gastrocnemius muscles from WT animals treatedwith FST(291)-IgG2 were significantly greater in size compared to thecontralateral legs as well as the vehicle controls (P<0.001). In bothstudies, the gastrocnemius muscle treated with FST(291)-IgG2 wassignificantly greater in size, normalized to body weight, compared tothe contralateral muscle and to vehicle-treated animals, in both WT andmdx mice. The increase in muscle mass was somewhat more pronounced inyounger animals than in older animals. In terms of percent increaserelative to the contralateral muscle, FST(291)-IgG2 increased musclemass by 34.2% and 16.4% in 6-week old WT and mdx mice, respectively.Muscle mass increases of 62.8% and 41.8% were observed in 4-week old WTand mdx mice, respectively.

These data demonstrate that blocking activin/myostatin signaling usingtwice per week intramuscular FST(291)-IgG2 administration increasesmuscle mass in a mouse model of a muscular dystrophy. The increase inmuscle mass occurs locally in the injected muscle only.

Example 9: The Effect of Local Administration of FST(291)-IgG2 Proteinon Muscle in a Mouse Model of ALS

The effect of FST(291)-IgG2 on muscle mass and strength was assessed ina mouse model of ALS. The B6.Cg-Tg(SOD1*G93A)1Gura (SOD1) strain of miceis a well-established model of human ALS (Wooley C M. et al. (2005)Muscle Nerve 32(1):43-50).

Twelve week old SOD1 mice and wild-type background strain B6 (WT) micereceived treatment with 100 μg of FST(291)-IgG2 or vehicle controlintramuscularly into the right tibialis anterior (TA) muscle, twice perweek. After 4 weeks, mice were anesthetized and the TA muscle wasprepared for physiological analysis, after which the injected anduninjected muscles were weighed and then processed for histologicalanalysis. Physiological strength analysis was performed by fixing thedistal TA tendon to a lever arm of a dual mode muscle lever system andinserting platinum needle electrodes behind the knee to stimulate theperoneal nerve.

In WT animals, FST(291)-IgG2 treatment increased TA muscle mass by 75%compared to the non-injected contralateral TA. The adjacent extensordigitorum longus (EDL) was also increased in mass by 118% compared tothe non-injected contralateral EDL. SOD1 mice displayed approximately50% less mass than WT mice. FST(291)-IgG2 treatment increased TA musclemass by 35% and EDL muscle mass 32% compared to the non-injectedcontralateral muscles.

In physiological strength tests of TA muscle, SOD1 mice displayed adeficit in strength compared to WT mice. TA muscle from vehicle controltreated SOD1 mice had a peak tetanic force of only 18% of the WT vehiclecontrol treated TA muscle. In WT mice, FST(291)-IgG2 treatment increasedthe peak tetanic force of the TA by 36% compared to vehicle controltreated WT mice. Moreover, there was a 72% increase in peak tetanicforce of the TA from FST(291)-IgG2 treated SOD1 mice compared to vehiclecontrol treated SOD1 mice.

These results demonstrate that FST(291)-IgG2 treatment can effectivelyincrease muscle mass and strength locally following direct injectioninto target muscles in both normal and neurogenic muscles. SOD1 micelose 50% of the muscle mass and 82% of the peak tetanic force in theirTA compared to wild type B6 mice. FST(291)-IgG2 increased both musclemass (35%) and peak tetanic force (72%) in SOD1 mice. These datademonstrate that FST(291)-IgG2 is a promising treatment for focalmyopathies and can improve muscle function in neurogenic diseases suchas ALS.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1. A method of treating Duchenne Muscular Dystrophy (DMD) in a patient, the method comprising: administering an effective amount of a follistatin polypeptide a patient in need thereof; wherein the follistatin fusion protein comprises a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 15 or 16, wherein the first amino acid sequence ends at the amino acid corresponding to any one of amino acids 291-302 of SEQ ID NO: 4, and wherein the second amino acid sequence comprises a constant domain of an immunoglobulin G (IgG).
 2. The method of claim 1, wherein administration of the effective amount increases muscle mass in a targeted muscle of the patient.
 3. The method of claim 1, wherein the follistatin polypeptide does not have a substantial systemic effect on muscle mass of the patient.
 4. The method of claim 2, wherein the targeted muscle is damaged, weakened or deficient.
 5. The method of claim 2, wherein the targeted muscle is healthy but an increase in muscle size or strength of the targeted muscle is desired. 6-8. (canceled)
 9. The method of claim 1, wherein the follistatin polypeptide binds to one or more ligands selected from the group consisting of: myostatin, GDF-11, activin A and activin B with a KD less than 1 nM, 100 pM, 50 pM or 10 pM. 10-12. (canceled)
 13. The method of claim 9, wherein the follistatin polypeptide comprises an unmasked heparin binding domain. 14-17. (canceled)
 18. The method of claim 13, wherein the follistatin polypeptide forms a dimer.
 19. (canceled)
 20. The method of claim 18, wherein the follistatin polypeptide comprises an Fc portion of an IgG.
 21. The method of claim 20, wherein the IgG is selected from the group consisting of IgG1, IgG2, IgG4 and an IgG2/4 hybrid. 22-24. (canceled)
 25. The method of claim 21, wherein the follistatin polypeptide comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:
 43. 26. The method of claim 25, wherein the follistatin polypeptide comprises the amino acid sequence of SEQ ID NO:
 43. 27. (canceled)
 28. The method of claim 26, wherein final (carboxy-terminal) lysine (K) of SEQ ID NO: 43 is absent.
 29. The method of claim 21, wherein the follistatin polypeptide comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:
 42. 30. The method of claim 29, wherein the follistatin polypeptide comprises the amino acid sequence of SEQ ID NO:
 42. 31. (canceled)
 32. The method of claim 30, wherein final (carboxy-terminal) lysine (K) of SEQ ID NO: 42 is absent.
 33. The method of claim 21, wherein the follistatin polypeptide comprises a first amino acid sequence derived from follistatin that consists of an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 15 or
 16. 34. (canceled)
 35. The method of claim 33, wherein the follistatin polypeptide comprises the amino acid sequence of SEQ ID NO:
 15. 36-37. (canceled)
 38. The method of claim 33, wherein the follistatin polypeptide comprises the amino acid sequence of SEQ ID NO:
 16. 39-45. (canceled)
 46. The method of claim 33, wherein the method causes no substantial effect in the patient on a measure selected from the group consisting of: serum FSH levels, liver size, hematocrit and reticulocyte levels. 